Citrus Huanglongbing, one of the most devastating citrus diseases, is caused by Candidatus Liberibacter asiaticus (CLas). Polyamines are aliphatic nitrogen-containing compounds that play important roles in disease resistance. However, the role of polyamine metabolism in the tolerance of citrus to infection with CLas have not been extensively studied. We used HPLC and UPLC-Q/TOF-MS to detect the contents of nine polyamine metabolism-related compounds (PMRCs) in six citrus cultivars with varying levels of tolerance to CLas. Moreover, we systematically detected the changes of PMRC and H2O2 contents, and compared the gene expression levels and activities of enzymes involved in the polyamine metabolic pathway among healthy, asymptomatic, and symptomatic leaves of Newhall navel oranges infected with CLas. The tolerant and moderately tolerant varieties showed higher PMRC levels than those of susceptible varieties. Compared with the healthy group, the symptomatic group showed significantly increased contents of arginine, ornithine, γ-aminobutyric acid, and putrescine by approximately 180, 19, 1.5, and 0.2 times, respectively, and up-regulated expression of biosynthetic genes. Arginase and ornithine decarboxylase enzyme activities were the highest in the symptomatic group, while arginine decarboxylase and agmatine deiminase enzyme activities were the highest in the asymptomatic group. The two polyamine biosynthetic pathways showed different trends with the increase of the titer of CLas, indicating that polyamines were mainly synthesized through the arginine decarboxylase pathway in the asymptomatic leaves, and were synthesized via the ornithine decarboxylase pathway in symptomatic leaves. These findings provide new insight into the changes of polyamine metabolism in citrus infected with CLas.